CN105315362A - Method for the preparation of human albumin with reduced level of dissolved oxygen - Google Patents
Method for the preparation of human albumin with reduced level of dissolved oxygen Download PDFInfo
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- CN105315362A CN105315362A CN201510264800.9A CN201510264800A CN105315362A CN 105315362 A CN105315362 A CN 105315362A CN 201510264800 A CN201510264800 A CN 201510264800A CN 105315362 A CN105315362 A CN 105315362A
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- 238000000034 method Methods 0.000 title claims abstract description 76
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 239000001301 oxygen Substances 0.000 title claims abstract description 43
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 43
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 18
- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 102000009027 Albumins Human genes 0.000 claims abstract description 91
- 108010088751 Albumins Proteins 0.000 claims abstract description 91
- 230000009467 reduction Effects 0.000 claims abstract description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 59
- 238000009928 pasteurization Methods 0.000 claims description 33
- 229910052757 nitrogen Inorganic materials 0.000 claims description 30
- 210000002381 plasma Anatomy 0.000 claims description 15
- 239000001307 helium Substances 0.000 claims description 12
- 229910052734 helium Inorganic materials 0.000 claims description 12
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 12
- 238000004381 surface treatment Methods 0.000 claims description 8
- 239000007789 gas Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000011261 inert gas Substances 0.000 claims description 6
- 239000012298 atmosphere Substances 0.000 claims description 5
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- 239000000463 material Substances 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
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- 230000009261 transgenic effect Effects 0.000 claims 1
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- 239000000523 sample Substances 0.000 description 28
- 230000003647 oxidation Effects 0.000 description 16
- 238000007254 oxidation reaction Methods 0.000 description 16
- 239000000047 product Substances 0.000 description 15
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- 230000008569 process Effects 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 8
- 239000002033 PVDF binder Substances 0.000 description 6
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 6
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 5
- 238000011026 diafiltration Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000011146 sterile filtration Methods 0.000 description 5
- 238000005571 anion exchange chromatography Methods 0.000 description 4
- 238000012859 sterile filling Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 238000007669 thermal treatment Methods 0.000 description 4
- 239000008215 water for injection Substances 0.000 description 4
- 102000004506 Blood Proteins Human genes 0.000 description 3
- 108010017384 Blood Proteins Proteins 0.000 description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- 206010020852 Hypertonia Diseases 0.000 description 3
- 229920005556 chlorobutyl Polymers 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000008174 sterile solution Substances 0.000 description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 2
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- 108010059904 nonmercaptalbumin Proteins 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- ZUKPVRWZDMRIEO-VKHMYHEASA-N L-cysteinylglycine Chemical group SC[C@H]([NH3+])C(=O)NCC([O-])=O ZUKPVRWZDMRIEO-VKHMYHEASA-N 0.000 description 1
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 1
- DZTHIGRZJZPRDV-LBPRGKRZSA-N N-acetyl-L-tryptophan Chemical compound C1=CC=C2C(C[C@H](NC(=O)C)C(O)=O)=CNC2=C1 DZTHIGRZJZPRDV-LBPRGKRZSA-N 0.000 description 1
- DZTHIGRZJZPRDV-UHFFFAOYSA-N Nalpha-Acetyltryptophan Natural products C1=CC=C2C(CC(NC(=O)C)C(O)=O)=CNC2=C1 DZTHIGRZJZPRDV-UHFFFAOYSA-N 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
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- 210000004369 blood Anatomy 0.000 description 1
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- 238000013375 chromatographic separation Methods 0.000 description 1
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- 208000031513 cyst Diseases 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
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- 230000005284 excitation Effects 0.000 description 1
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- 239000012530 fluid Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000009593 lumbar puncture Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- -1 mercaptan compound Chemical class 0.000 description 1
- 108010004546 mercaptoalbumin Proteins 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229940116191 n-acetyltryptophan Drugs 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
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- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- BYKRNSHANADUFY-UHFFFAOYSA-M sodium octanoate Chemical compound [Na+].CCCCCCCC([O-])=O BYKRNSHANADUFY-UHFFFAOYSA-M 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- General Health & Medical Sciences (AREA)
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- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
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- Engineering & Computer Science (AREA)
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- Inorganic Chemistry (AREA)
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- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a method for the preparation of a solution of human albumin, more particularly it relates to a method comprising a stage of reduction of the dissolved oxygen in said solution of albumin until a concentration equal to or less than 0.5 ppm. With the method of the present invention it is possible to obtain a solution of human albumin having a redox state closer to the redox state of the albumin present in human plasma.
Description
describe
The present invention relates to the method for the preparation of human albumin solution, more specifically, the present invention relates to a kind of method, described method comprises and reduces dissolved oxygen in described albumin solution until be equal to or less than the step of the concentration of 0.5ppm.With the inventive method, obtain have with the albuminous redox state existed in human plasma closer to the human albumin solution of redox state be possible.
Human albumin is the nonglycosylated albumen of 66kDa.Quantitatively, it is most important plasma proteins and its concentration is between 35g/l and 50g/l in normal plasma, and what form total plasma proteins reaches 60% (PetersT.J.:AllAboutAlbumin; Biochemistry, GeneticsandMedicalApplications.AcademicPress, SanDiego, 1996).
The structure of human albumin forms by having 585 amino acid whose polypeptide, there is about 67% α spiral, not there is β-pleated sheet structure and there is (OtagiriM., ChuangV.T.:Pharmaceuticallyimportantpre-andposttranslati onalmodificationsonhumanserumalbumin.BioPharmBull2009,32 phases, 527-534 page).Human albumin comprises 6 methionine residues and 35 cysteine residues of the formation of participation 17 disulfide linkage.Cys-34 is unique free halfcystine in whole molecule.Human albumin is captured (radicalentrapment) characteristic and is had specific anti-oxidant function due to the ability that combines with multiligand (multipleligands) and its free radical, all relevant with its close structure with its both free radical entrapment properties to the ability that multiligand combines.
Although there is the albuminous many possibilities of oxidation, Cys-34 is the site responsive especially to oxidation/reduction.Therefore, usually, it is rational in Cyst-34, referring to albuminous redox state.According to the redox state of Cys-34, by albuminous chromatographic separation, obtain three kinds of fractions (Peters, 1996, the same):
(i) reduction form, wherein halfcystine is with the form of free thiol group, is called as people's mercaptalbumin (humanmercaptoalbumin, HMA);
(ii) oxidised form, wherein halfcystine and little mercaptan compound, mainly halfcystine or cysteinyl glycine form disulfide linkage, although also form disulfide linkage with homocysteine and gsh, be called as the non-mercaptalbumin 1 (humannon-mercaptoalbumin1, HNA1) of people; And
(iii) highest oxidation form, wherein halfcystine is-sulfinic acid or sulfonic acid, is called as the non-mercaptalbumin 2 (humannon-mercaptoalbumin2, HNA2) of people.
In Healthy People, total albumin of about 50-69% is the form of HMA, 27-42% is the form of HNA1, and 3-5% is the form (OettlK. of HNA2, Deng people Oxidativedamageofalbumininadvancedliverdisease.Biochim.B iophys.Acta2008,1782 phases, 469-473 page; OettlK. with MarscheG.RedoxStateofhumanserumalbuminintermsofcysteine-34inhealthanddisease.MethodsEnzymol.2010,474 phases, 181-195 page; And OettlK., wait people Oxidativealbumindamageinchronicliverfailure:Relationtoal buminbindingcapacity; Liverdysfunctionandsurvival.JHepatol, 2013,59 phases, 978-983 page).Usually it is believed that HMA is oxidized to HNA1 is reversible process, and HMA is oxidized to HNA2 is irreversible process.
Albumin during the side that uses, can experience the amendment of various structures, cause the amendment of its conformation in vivo and producing therapeutic albumin, and as a result, the amendment of binding characteristic together with its redox state (people such as Oettl, K., 2010, the same).
Normally used method in the fractional separation of the human plasma of the albumen for obtaining purifying, wherein be used to find that albuminous is the Cohn method (people such as CohnE.J., ' Preparationandpropertiesofserumplasmaproteins, IV.Asystemfortheseparationintofractionsoftheproteinandli poproteincomponentsofbiologicaltissuesandfluids.J.Am.Che m.Soc.1946,68 phases, 459-475 page), or its less amendment.
Cohn method starts with human plasma, precipitates and the sequential step be separated it.In respective step, obtain the throw out and the supernatant decanted liquid (decantationsupernatant) that are rich in one of plasma proteins.For realizing precipitation Cohn fraction (fraction I, fraction II+III, fraction IV and fraction V) in succession, to change the object of the solubleness of different proteins, need the parameter revising solution, such as, especially, pH, specific inductivity, temperature, protein concn and ionic strength.In addition, it should be noted that described Cohn fraction comprises the ethanol of progressive concentration.As a result, the albumin be included in the supernatant liquor of fraction IV is precipitated with about 40% (v/v) ethanol, and continues the part of the slurry (paste) forming fraction V.
Acquisition fraction V after, by the latter in the solution resuspended and different step is carried out to it until obtain final product.Habitually, these steps comprise: the final pasteurization of diafiltration, thermal treatment, sterilizing, loading bottle and described bottle, usual during 30-32 DEG C is no less than the time period of 14 days subsequently, submit to described bottle to quarantine (quarantine), object is the aseptic of guarantee the finished product.
The present inventor have been found that from human plasma obtain the process of albumin solution during, albumin suffers the amendment of the redox state to Cys-34.These amendments fundamentally occur between the shelf lives of oxygen existence, and they are detected according to quarantine step in essence by this.In some production batchs (n=7), the level of HMA, HNA1 and HNA2 that has been found that is 40-53%, 39-44% and 7-16% (w/v) respectively, and result, the level of HMA and HNA2 is mainly different from those (OettlK.2008,2010 and 2013, the same) of describing in Healthy People.This due to, such as, in the situation of HNA2, the oxidation to HNA2 as hereinbefore set forth is the fact of irreversible process, and can be significantly important.
Unexpectedly, contriver has been found that, by producing the step adding in human albumin solution's process and comprise the dissolved oxygen reduced in solution, wherein the level of oxygen is reduced to the concentration being equal to or less than 0.5ppm, realize the reduction of the oxidation of Cys-34, the albuminous redox state of acquisition is closely similar with the albuminous redox state existed in blood plasma.This causes the following fact: the albuminous characteristic of acquisition, and such as, its anti-oxidation characteristics, more similar to those characteristics albuminous existed in blood, this can cause the improvement of its therapeutic efficiency in its many application.
Therefore, present invention is disclosed the method for the preparation of human albumin solution, it is characterized in that it comprises the step reducing dissolved oxygen in described albumin solution, the level of wherein said oxygen is reduced to the concentration being equal to or less than 0.5ppm.Preferably, after the step reducing dissolved oxygen in albumin solution, described albumin solution is maintained in inert gas atmosphere.
In described reduction albumin solution, the step of dissolved oxygen can be carried out with the various ways that this area is known at present.Preferably, the surface treatment of albumin solution can realize with rare gas element or rare gas element can be entered the inside of described albumin solution by bubbling.The described rare gas element used in the method for the invention can be nitrogen, helium or similar gas.
Method of the present invention can be used to the albumin solution that acquisition has the albumin concentration between about 4% and 25% (w/v).Preferably, the albumin of acquisition is therapeutic albumin.
In addition, albumin of the present invention can be the albumin of form or the genetically modified form acquisition of recombinating.Albumin or the genetically modified albuminous molecule of restructuring are identical with human albumin in its amino acid whose sequence, it there is not glycosylation and, be functional object with it, it must show that the conformation identical with the albumin that human plasma is originated is folding.If not so, except other possibility side reactions caused by described difference, due to immunogenic danger, it can not be used by people.
The step reducing the dissolved oxygen in albumin solution of the present invention can be implemented before or after the pasteurising step of described albumin solution, or in addition, although the pasteurising step of described albumin solution is not implemented, it can not rely on the process of the initial albumin solution of preparation yet and is performed.
For obtaining the better result in redox state aspect of Cys-34 in the albumin solution that obtained by method of the present invention, preferably, after the step reducing dissolved oxygen in albumin solution, described albumin solution is maintained in inert gas atmosphere.Described inert gas atmosphere can be nitrogen, helium or similar gas.
Although utilize the packaged and/or any container being stored in wherein in those of the albumin obtained by method of the present invention to be possible, preferably described container makes by the impervious material of oxygen, is more preferably made up of glass.
Another object of the present invention discloses composition and its purposes as medicine, and described composition comprises the human albumin prepared by method of the present invention.
Finally, present invention is disclosed and comprise the albuminous composition prepared according to the method that above the describes purposes for the preparation of medicine.
The present invention is described in more detail below about embodiment and comparing embodiment, and described embodiment and comparing embodiment do not form restriction of the present invention.In addition, reference is carried out to accompanying drawing, wherein:
Fig. 1 shows the schematic diagram obtaining the method for therapeutic human albumin from blood plasma utilized in the prior art.
Fig. 2 shows the figure of the concentration of HMA, HNA1 and HNA2 form of albuminous Cys-34, is expressed as the height at the peak obtained by red, orange, green, blue, yellow (ROGBY) in multiple steps of the method utilized in prior art before making the present invention: () is from the blood plasma (n=59) of healthy donors; (★) from the supernatant liquor (n=3) of cryoprecipitation;
albumin solution (n=3) before stablizer adds and before thermal treatment;
there is the albumin solution (n=1) before stablizer and thermal treatment; (■) there is the albumin solution (n=1) after stablizer and thermal treatment; 20% albumin solution (n=3) before (◆) sterile filtration; (▲) aseptic 20% albumin solution and in final container (n=4);
in final container and 20% albumin solution (n=4) of the pasteurization of not quarantining; (zero) final 20% Albumin products (n=7); Wherein n represent analysis batch number.
Fig. 3 shows the figure of the concentration of HMA, HNA1 and HNA2 form of albuminous Cys-34, the height at the peak that the method being expressed as the prior art before utilizing the present invention is obtained by red, orange, green, blue, yellow (ROGBY) in multiple steps:
25% aseptic albumin solution and in final container (n=1);
25% albumin solution (n=1) of not quarantining of pasteurization; With
25% final Albumin products (n=1);
5% aseptic albumin solution and in final container (n=1);
in final container and 5% albumin solution (n=1) of the pasteurization of not quarantining; And
final 5% Albumin products (n=1); Wherein n represent analysis batch number.
Fig. 4 shows the figure of the concentration of HMA, HNA1 and HNA2 form of albuminous Cys-34, be expressed as the height at the peak utilizing method of the present invention to be obtained by red, orange, green, blue, yellow (ROGBY) in multiple steps, wherein used with gaseous nitrogen surface-treated step before pasteurization: 20% albumin solution that (▲) is aseptic and in final container (n=3);
in final container and 20% albumin solution (n=3) of the pasteurization of not quarantining; (zero) final 20% Albumin products (n=3); Wherein n represent analysis batch number.
Fig. 5 shows the figure of the concentration of HMA, HNA1 and HNA2 form of albuminous Cys-34, be expressed as the height at the peak utilizing method of the present invention to be obtained by red, orange, green, blue, yellow (ROGBY) in multiple steps, wherein before pasteurization, use the step of process gaseous nitrogen bubbling being entered albumin solution: 20% albumin solution that (▲) is aseptic and in final container (n=3);
in final container and 20% albumin solution (n=3) of the pasteurization of not quarantining; (zero) final 20% Albumin products (n=3); Wherein n represent analysis batch number.
Fig. 6 shows the figure of the concentration of HMA, HNA1 and HNA2 form of albuminous Cys-34, be expressed as the height at the peak utilizing method of the present invention to be obtained by red, orange, green, blue, yellow (ROGBY) in multiple steps, wherein used with gaseous nitrogen surface-treated step before pasteurization:
unpacked aseptic 25% albumin solution (n=1);
pasteurization and 25% albumin solution (n=1) of not quarantining; With
final 25% Albumin products (n=1);
5% aseptic albumin solution and in final container (n=1);
in final container and 5% albumin solution (n=1) of the pasteurization of not quarantining; And
final 5% Albumin products (n=1); Wherein n represent analysis batch number.
Fig. 7 shows the figure of the concentration of HMA, HNA1 and HNA2 form of albuminous Cys-34, be expressed as the height at the peak utilizing method of the present invention to be obtained by red, orange, green, blue, yellow (ROGBY) in multiple steps, wherein used with gaseous helium surface-treated step before pasteurization:
25% aseptic albumin solution and in final container (n=1);
in final container and 25% albumin solution (n=1) of the pasteurization of not quarantining; With
25% final Albumin products; Wherein n represent analysis batch number.
Embodiment
Embodiment 1: according to prior art for obtaining albuminous method
According to the method (people such as CohnE.J. of Cohn, 1946, ditto) human plasma obtained from healthy donors is precipitated and the step be in succession separated, from obtaining initial freezing precipitation supernatant liquor until realize the precipitation (see Fig. 1) of fraction V.The fraction V of Cohn is suspended in cold water for injection (waterforinjection, WFI), it is adjusted to pH7.0 and is clarified by deep filter (in-depthfilter).Clear soln, applies the dialysis solution and holding temperature that are formed by the salt of monovalent ion (sodium-chlor) at 5 DEG C, with constant volume by diafiltration (diafilter) (Fig. 1, clarification/diafiltration steps).Sodium octoate and N-acetyltryptophan are added to the solution of diafiltration as stablizer.Described solution is carried out at 60 DEG C of heat treated (Fig. 1, heat treatment step).Subsequently, the function (such as, 5%, 20% or 25% (w/v)) (Fig. 1, unpacked solution) of the concentration of the expectation of the final protein that heat treated solution WFI dilutes or simmer down to is expected.Then final solution filters in aseptic mode (0.22 μm of strainer) and carries out the filling (Fig. 1, sterile filtration and filling step) of final sterile chamber in aseptic area.Solution in final container is no less than 10h (Fig. 1, in the vial pasteurising step) 60 DEG C of heating.Finally, bottle is hatched at 30-32 DEG C and is no less than 14 days (Fig. 1, quarantine step).After the described time period, bottle by visual inspection to discard the indication (Fig. 1, final Albumin products) of any microbial contamination.
Based on by OettlK., 2010, the same method that describes and as described in detail later, the oxidation state (Fig. 2) of albumin sample from the different step obtaining albuminous process is analyzed by high performance liquid chromatography (HPLC).
Albuminous diluted sample is under study for action become the concentration of 6.5mg/ml in the damping fluid of 0.3M sodium-chlor, 0.1M sodium phosphate pH6.87, and 5 μ l samples are entered ShodexAsahipakES-502NDEAE anion-exchange column (7.5 × 100mm with the injection of 1.0ml/min flow velocity, Shodex, Japan).According to utilizing 50mM sodium acetate and 400mM sodium sulphate gradient, its wash-out is performed at pH4.85, until be issued to 6% ethanol at 35 DEG C at 1.0ml/min constant flow rate, realize, according to its oxidation state, albumin sample is separated into three kinds of fractions (HMA, HNA1 and HNA2).
First 5 minutes of wash-out carry out not existing under ethanol.At ensuing 5-35 minute, the concentration of ethanol is increased to 6% in a linear fashion, maintains that it is constant subsequently other 5 minutes periods.Finally, from the 40 to 45 minute, the concentration of ethanol is down to 0% again.There is no ethanol again after 5 minutes, next sample can be analyzed.
Utilize excitation wavelength and the emission wavelength of 280nm and 340nm respectively, the detection of three kinds of albumin fraction as the function of its oxidation state is undertaken by fluorescence.Consider the height at each peak interested obtained in the color atlas of correspondence and carry out the quantitative of the concentration of HMA, HNA1 and HNA2 form of albuminous Cys-34.
Fig. 2 shows during the albuminous process obtaining prior art, from the change of the oxidation state of the sample of the human serum albumin of different step.The rising of data presentation HNA1 and HNA2 form is unfavorable for HMA form, especially after with pasteurization, the step of quarantining subsequently (20% final Albumin products).With in 5% and 25% albumin concentration of the method for prior art from the purifying of human plasma, the equivalent performance (Fig. 2) that 20% final Albumin products after identical technology is observed of performance (Fig. 3) of the albuminous oxidation state after the step of pasteurization and quarantine.In both figures, the data presentation HNA1 of acquisition and the rising of HNA2 form are unfavorable for HMA form, especially, after with pasteurization, the step of quarantining subsequently.
Embodiment 2: utilize and utilized with nitrogen surface-treated step acquisition albuminous method of the present invention before pasteurization.
Corresponding with the method described in embodiment 1 for obtaining albuminous method of the present invention, also comprise the step reducing dissolved oxygen in albumin solution, as described below.
(Fig. 1 obtain sterile solution in final container after, sterile filtration and filling step), and with the object of the oxygen of the inside being substituted in container existence, carry out the surface treatment with nitrogen, by two hypodermic needles (be business type, BraunSterican21Gx1
1/
2"; 0.80x40mm, Germany, or similar) chlorinated butyl rubber bung of insertion bottle; described hypodermic needle is connected to two 0.22 μm of PVDF strainers (be business type; MillexGVMillipore, 0.22 μm, PVDF; 13mm strainer; the U.S., or similar), avoids pin to contact with albumin solution.One of pin is pre the entrance of nitrogen and another is its outlet, has the object preventing hypertonia in container.At room temperature perform two hours with surface nitrogen gas disposal, maintain the steady flow of nitrogen, have and allow to observe liquid in containers motion, and the object that it does not splash in a reservoir.
After completing the surface treatment with nitrogen, continue to obtain albuminous method (Fig. 1, in the vial pasteurising step), until obtain final Albumin products as what describe in embodiment 1 from human plasma.In mode identical in such as embodiment 1, analyzed the oxidation state of 20% concentration samples obtained with technology of the present invention by anion-exchange chromatography.Particularly, analyze experience before pasteurization with the albuminous sample of nitrogen surface-treated step, after pasteurization with quarantine before albuminous sample and quarantine the time period after albuminous sample.Fig. 4 shows the result of acquisition, observe with prior art for obtaining albuminous method, in figs. 2 and 3, HMA form does not reduce, HNA1 and HNA2 form does not raise.
Except the analysis of oxidation state, carry out the dissolved oxygen existed in measure sample after with nitrogen surface treatment, result is that the concentration of dissolved oxygen is in all cases equal to or less than 0.5ppm.
Measure and at room temperature carry out for the probe (being business type, HI9828MultiparameterMeter, HannaInstruments, the U.S.) measuring dissolved oxygen by using.Particularly, have to experience in advance and open with the inside of the container of nitrogen surface-treated albumin solution at the booth (cubicle) with nitrogen atmosphere, and immersed immediately for the probe of oxygen in measure sample.The probe used, its function, based on the principle of galvanic cell, comprises silver (Ag) positive pole, and described silver positive electrode cover has function to be platinum (Pt) line of negative pole.Aforementioned assemblage is injected the shield cap being full of Repone K electrolytic solution, it has at its end
film, a kind of oxygen existed in the solution that allows passes through, but do not allow solution self to pass through can through the material of oxygen.By applying 790mV electromotive force, the oxygen existed in cell is reduced to hydroxide ion (OH at negative electrode
-), and silver chloride precipitates at positive pole.This reaction brings the electric current with the intensity proportional with the amount of oxygen that exists in sample.Then the current conversion measured is become the concentration of corresponding dissolved oxygen by instrument.
Embodiment 3: utilize step nitrogen bubble being entered solution before pasteurization to obtain albuminous method of the present invention.
Corresponding with the method described in embodiment 1 for obtaining albuminous method of the present invention, and comprise step described below.Obtain sterile solution in final container after (Fig. 1, sterile filtration and filling step), and with the object of oxygen that the inside being substituted in container exists, by by two hypodermic needles (be business type, BraunSterican21Gx1
1/
2"; 0.80x40mm; Germany, or similar) chlorinated butyl rubber bung of insertion bottle performs the process of nitrogen bubble, and described hypodermic needle is connected to two 0.22 μm of PVDF strainers (being business type; MillexGVMillipore; 0.22 μm, PVDF, 13mm strainer; the U.S., or similar).Lancet is entered by what be pre nitrogen, and lumbar puncture needle (be business type, TerumoSpinalNeedle, 18Gx3
1/
2", 1.20x90mm, Japan, or similar), immerse in albumin solution, and by avoid with liquid comes into contact and locate hypodermic needle (be business type, BraunSterican21Gx1
1/
2", 0.80x40mm, Germany, or similar), be appointed as its outlet in advance, there is the object preventing hypertonia in a reservoir.The process of nitrogen bubble is at room temperature performed two hours, maintains the steady flow of nitrogen, there is the object allowing to observe small bubbles in liquid.
After completing the process by nitrogen bubble, continue as describe in embodiment 1 for obtaining albuminous method (Fig. 1, in the vial pasteurising step) from human plasma, until obtain final Albumin products.
In mode identical in such as embodiment 1 and embodiment 2, the anion-exchange chromatography of the sample of 20% albumin concentration obtained by technology of the present invention analyzes oxidation state.Particularly, analyze to experience before the step of pasteurization by the albuminous sample of the step of nitrogen bubble, after pasteurization with quarantine before albuminous sample and albumin sample after the quarantine time period.Fig. 5 shows the result of acquisition, and these are similar with utilizing before pasteurization with those results obtained in Fig. 4 of nitrogen surface-treated step.Observe with prior art for obtaining albuminous method, in figs. 2 and 3, HMA form does not reduce, HNA1 and HNA2 form does not raise.
In this case, carry out the measurement of the dissolved oxygen existed in sample after by nitrogen bubble process in the identical mode in such as embodiment 2, result is in all cases, and the concentration of dissolved oxygen is equal to or less than 0.5ppm.
Embodiment 4: utilize with nitrogen surface-treated step before pasteurization for obtaining albuminous method of the present invention, be applied to the concentration that albuminous difference is final.
Corresponding with the method described in example 2 for obtaining albuminous method of the present invention, be applied to such as, 5 and 25% other concentration albuminous.In mode identical in such as embodiment 2, analyzed 5 and 25% oxidation state of albuminous sample of concentration that obtain with the technology of the present invention by anion-exchange chromatography.Particularly, analyze experience before pasteurization with 5 of nitrogen surface-treated step and 25% albuminous sample, after pasteurization with quarantine before 5 and 25% albuminous sample and 5 after the quarantine time period and 25% albuminous sample.Fig. 6 shows the result of acquisition, these be similar for those results obtained in albuminous Fig. 4 of the concentration of 20%.Observe the albuminous method for obtaining prior art, in figs. 2 and 3, HMA form does not reduce, HNA1 and HNA2 form does not raise.
In addition, the measurement of the dissolved oxygen carry out the analysis of oxidation state, having in the sample after nitrogen surface treatment, result is that the concentration of dissolved oxygen is in all cases equal to or less than 0.5ppm.
Embodiment 5: to utilize before pasteurization with helium surface-treated step for obtaining albuminous method of the present invention.
Corresponding with the method described in embodiment 1 for obtaining albuminous method of the present invention, and comprise step described below.(Fig. 1 obtain sterile solution in final container after, sterile filtration and filling step), and with the object of the oxygen existed in replacement in the vial remaining wind box, carry out the surface treatment with helium, by two hypodermic needles (be business type, BraunSterican21Gx1
1/
2"; 0.80x40mm, Germany, or similar) insertion chlorinated butyl rubber bung; described hypodermic needle is connected to two 0.22 μm of PVDF strainers (be business type; MillexGVMillipore, 0.22 μm, PVDF; 13mm strainer; the U.S., or similar), avoids pin to contact with albumin solution.One of pin is pre the entrance of helium and another is its outlet, has the object preventing hypertonia in a reservoir.At room temperature perform two hours with surperficial helium process, maintain the steady flow of nitrogen, there is the object allowing to observe liquid in containers motion, and it does not splash in a reservoir.
After completing the surface treatment with helium, continue as describe in embodiment 1 for obtaining albuminous method (Fig. 1, in the vial pasteurising step) from human plasma, until obtain final Albumin products.
In mode identical in such as embodiment 1 to 4, analyzed the oxidation state of the albuminous sample obtained with technology of the present invention by anion-exchange chromatography.Particularly, analyze to experience before pasteurization with the albuminous sample of helium surface-treated step, after pasteurization with quarantine before albuminous sample and albuminous sample after the quarantine time period.Fig. 7 shows the result of acquisition, these results with utilize those results obtained in Fig. 4 and Fig. 6 with nitrogen surface-treated step before pasteurization to be similar, and also nitrogen bubble to be entered with utilization those results obtained in Fig. 5 of the step of solution before pasteurization be similar.Observe with prior art for obtaining albuminous method, in figs. 2 and 3, HMA form does not reduce, HNA1 and HNA2 form does not raise.
In the case, carry out the dissolved oxygen existed in measure sample after the process by bubbling helium in same mode in such as embodiment 2, result is that the concentration of dissolved oxygen is in all cases equal to or less than 0.5ppm.
Claims (14)
1., for the preparation of the method for human albumin solution, it is characterized in that described method comprises the step reducing dissolved oxygen in described albumin solution, the level of wherein said oxygen is reduced to the concentration being equal to or less than 0.5ppm.
2. method according to claim 1, is characterized in that the step of dissolved oxygen in the described albumin solution of described reduction is by carrying out with albumin solution described in rare gas element surface treatment.
3. method according to claim 1, is characterized in that the step of dissolved oxygen in the described albumin solution of described reduction is undertaken by inside bubbling inert gas being entered described albumin solution.
4. the method according to any one in claims 1 to 3, is characterized in that described albumin is blood plasma recombinant sources or transgenic sources.
5. the method according to any one in Claims 1-4, is characterized in that described albumin solution has the albuminous concentration between about 4% and 25% (w/v).
6. the method according to any one in claim 2 to 5, is characterized in that used rare gas element is nitrogen or helium.
7. the method according to any one in claim 1 to 6, it is characterized in that the step of dissolved oxygen in the described albumin solution of described reduction the pasteurization of described albumin solution step come to carry out.
8. the method according to any one in claim 1 to 7, is characterized in that afterwards the carrying out of step of the step of dissolved oxygen described in the described albumin solution of described reduction at the pasteurization of described albumin solution.
9. the method according to any one in claim 1 to 8, after it is characterized in that the step of dissolved oxygen in the described albumin solution of described reduction, described albumin solution is maintained in inert gas atmosphere.
10. method according to claim 9, is characterized in that described inert gas atmosphere is nitrogen or helium.
11. methods according to any one in claim 1 to 10, is characterized in that described albumin solution is packaged and/or are stored in by the container made the impervious material of oxygen.
12. methods according to claim 11, is characterized in that described is glass to the impervious material of oxygen.
13. compositions, described composition comprises the human albumin by preparing according to the method for any one in claim 1 to 12, it is characterized in that described composition has the concentration of the dissolved oxygen being equal to or less than 0.5ppm.
14. according to claim 13ly comprise the purposes of albuminous composition for the preparation of medicine.
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| ES2900302T3 (en) | 2016-04-28 | 2022-03-16 | Alkahest Inc | Plasma fractions as therapy for tumor growth and progression |
| CA3033051A1 (en) | 2016-08-18 | 2018-02-22 | Alkahest, Inc. | Blood plasma fractions as a treatment for aging-associated cognitive disorders |
| BR112019022402A2 (en) | 2017-04-26 | 2020-05-19 | Alkahest Inc | dosing regimen to treat cognitive and motor impairments with blood plasma and blood plasma products |
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